Head and neck squamous cell carcinomas (HNSCCs) are a common human malignancy with 5-year survival rates that have not improved for the past several decades. The incidence of human papilloma virus (HPV)- associated HNSCC is increasing and represents an emerging health problem. Current therapies for HNSCC are associated with considerable toxicities and roughly 50% of patients suffer recurrence. Moreover, advanced stage or recurring HNSCC tumors are frequently chemoresistant. Our long-term goal is to develop novel therapeutic agents and strategies that can be used alone, or in combination with conventional treatments, to improve survival and reduce toxicities in HNSCC patients. Overexpression of anti-apoptotic members of the Bcl-2 protein family, including Bcl-XL and Bcl-2, is observed in a majority of HNSCC and correlates with chemotherapy resistance in this disease. In preliminary studies we have shown that a small molecule inhibitor of Bcl-XL and Bcl-2, ABT-737, synergized with cisplatin to kill HNSCC cells in vitro, via a process involving upregulation of pro-apoptotic Noxa. Additionally, the proteasome inhibitor bortezomib promoted HNSCC cell death in vitro, via induction of pro-apoptotic Bik and Bim, proteins that act as natural antagonists of Bcl-XL/Bcl-2. Bortezomib also induced molecular features of autophagy, and suppression of autophagy enhanced the in vitro resistance of HNSCC cells to bortezomib- induced cell death. The combination of bortezomib and cisplatin exhibited synergism in vitro and enhanced anti-tumor effects against HNSCC xenografts in vivo. HPV-positive HNSCC cells exhibited heighted sensitivity to bortezomib/chemotherapy, and preliminary findings suggest elevated levels of bortezomib-induced autophagy in HPV-positive cells. We hypothesize that HNSCC sensitivity to treatments that incorporate agents targeting the proteasome or anti-apoptotic Bcl-2 family members is modulated by autophagy, HPV, and induction of pro-apoptotic Bcl-2 family members. We propose three Specific Aims. Specific Aim 1 will investigate cellular mechanisms conferring in vitro sensitivity to proteasome inhibitor-based regimens, by examining the roles of autophagy induction and HPV. Specific Aim 2 will examine in vitro and in vivo anti- HNSCC effects, and corresponding mechanisms, resulting from targeting of anti-apoptotic Bcl-2 family members with the small molecule inhibitors ABT-737 and GX15-070, alone and in combination with chemotherapy. Specific Aim 3 will investigate in vivo anti-HNSCC tumor effects and mechanisms resulting from proteasome targeting, alone and in combination with targeting of anti-apoptotic Bcl-2 family members. We anticipate that results from our studies will elucidate unique mechanisms that control the sensitivities of HNSCC cells and tumors to agents targeting the proteasome or anti-apoptotic Bcl-2 family members. We also expect that our results will guide the design of novel treatment strategies and provide the basis for clinical evaluation of synergistic drug combinations in HNSCC.